We examined the reactivity of insulin receptor sulfhydryls to biotinylation in Chinese Hamster Ovary cells that express high levels of human insulin receptors (CHO/HIRc cells). Following the biotinylation reaction, the insulin receptor was purified by immunoprecipitation, and resolved by SDS-polyacrylamide gel electrophoresis before electrotransfer to membranes. The use of enzyme-linked streptavidin in conjunction with a chemiluminescent technique allowed the detection of thiol-biotinylated receptor beta-subunit, with no modification of the alpha-subunit. In cells expressing large numbers of IGF-1 receptors, the same technique enabled the detection of thiol-biotinylated IGF-1 receptors as well. Thiol-alkylation of intact CHO/HIRc cells with an impermeant reagent did not impair the ability of maleimidodibutyryl biocytin (MBB) to biotinylate sulfhydryls on the receptor beta-subunit after cell permeabilization with digitonin. In contrast, thiol-alkylation of digitonin-permeabilized cells prevented MBB-induced receptor biotinylation. The basal and insulin-activated insulin receptors exhibited insulin receptors exhibited a comparable reactivity to MBB. Furthermore, the use of affinity-purification on monomeric avidin-agarose enabled us to learn that the biotinylation reaction was near quantitative. MBB had no effect on insulin binding nor on receptor autophosphorylation and insulin-dependent receptor kinase activity. However, basal levels of receptor kinase activity were significantly elevated by thiol biotinylation. Further, in the presence of vanadate, MBB retained the ability to enhance receptor kinase activity in permeabilized cells, consistent with the notion that this increased exogenous substrate phosphorylation was not accounted for by inactivation of protein tyrosine phosphatases. The dephosphorylation of thiol-biotinylated, 32P- labeled insulin receptors by particulate protein tyrosine phosphatases was not affected. These results suggest that modification of reactive sulfhydryls located in the cytoplasmic domain of the insulin receptor beta-subunit may play a critical role in insulin receptor function.